利用CRISPR/Cas9技术编辑水稻ROP基因OsRac5

OsRac5基因属于水稻小G蛋白ROP家族。该基因参与了水稻的育性调节，但是OsRac5在水稻生长发育中的作用尚不清楚。为鉴定该基因的功能，本文采用CRISPR/Cas9基因组编辑技术，构建了该基因的双靶标载体Cas9-OsRac5，并对水稻进行了遗传转化。对转基因水稻的筛选和分子鉴定显示，在T1代获得了10个纯合突变株系。序列分析显示，在OsRac5编辑水稻中，该基因编码区发生了碱基缺失或/和插入，导致预期产生的不同类型OsRac5截短蛋白均丧失小G蛋白的保守结构域。对抽穗期OsRac5编辑水稻的表型进行统计学分析，结果显示，OsRac5编辑水稻与对照在剑叶角度以及剑叶净光合速率上存在极显著差异，其中OsRac5编辑水稻剑叶角度增大67%，剑叶净光合速率减小32.7%。本研究结果提示，OsRac5基因通过调控剑叶角度，影响水稻光合效率，与水稻生长发育密切相关。

Editing ROP Gene OsRac5 of Rice by CRISPR/Cas9 Technique

OsRac5 gene belongs to the small G protein ROP family of rice. The gene is involved in the regulation of rice fertility, but the role of OsRac5 in rice growth and development is still unclear. In order to identify the function of the gene, the double target vector Cas9-OsRac5, was constructed by CRISPR/Cas9 genome editing technique and the genetic transformation of rice was carried out. Screening and molecular identification of transgenic rice showed that 10 homozygous mutant lines were obtained in T1 generation. Sequence analysis showed that in OsRac5-edited rice, base deletion and/or insertion were occurred in the coding region of the gene resulting in the loss of conserved domains of small G proteins in the expected different types of OsRac5 truncated proteins. At heading stage, phenotype analysis statistically showed that there were significant differences in the flag leaf angle and net photosynthetic rate for OsRac5-edited rice and the control. The OsRac5-edited rice, the flag leaf angle increased by 67%, and the net photosynthetic rate of flag leaf decreased by 32.7%. Based on this study, it is suggested that OsRac5 gene affects photosynthetic efficiency of rice by regulating flag leaf angle, which is closely related to growth and development of rice.